Friend spleen focus-forming virus (SFFV)causes a rapid, multi-stage leukemia in mice and provides a model for understanding how retroviral envelope proteins can have pathogenic consequences. Our previous studies have shown that the envelope protein of Friend SFFV interacts with and activates a unique receptor tyrosine kinase, sf-Stk, which is preferentially expressed in erythroid cells. This results in constitutive activation of signal transduction pathways for the growth, differentiation and survival of erythroid cells. Some of these erythroid cells become transformed due to SFFV integration, which results in activation of the myeloid transcription factor PU.1 and a block in erythroid cell differentiation. We showed that this block was due to activation of the hematopoietic phosphatase SHP-1, which in turn causes dephosphorylation of STAT1, blocking its ability to bind DNA and activate genes associated with differentiation. We further demonstrated that these SFFV-transformed erythroleukemia (SFFV-MEL) cells can metastasize to the bone marrow, where they are retained and subsequently cause meningeal leukemia, a common neurological complication of human leukemia. Future work is aimed at better understanding the molecular basis for SFFV-induced meningeal leukemia and testing specific therapeutic strategies to block this retrovirus-induced multi-stage leukemia. In collaboration with Larry Keefer and members of his laboratory, we have shown that SFFV-MEL cells, but not normal hematopoietic cells, are killed by the nitric oxide prodrug JS-K. We demonstrated that JS-K induced necrosis and apoptosis in these cells by a caspase-dependent mechanism, causing DNA damage. We further demonstrated that JS-K treatment blocked the cell cycle by causing a block in the activation of the serine kinase Akt, leading to activation of the transcription factor FoxO3a, which in turn upregulated the cyclin-dependent kinase inhibitor p27. CDNB (1-chloro-2,4-dinitrobenzene), which contains an arylating ring analogous to that of JS-K without the diazeniumdiolate group necessary to release NO, was also able to kill SFFV-MEL cells by the same mechanisms, indicating that the arylating capability of JS-K, in addition to its ability to release NO, is a major contributor to the anticancer effects of this compound for SFFV-MEL cells. Studies are in progress to determine if JS-K can block the growth of SFFV-MEL cells in vivo. Several years ago we made the intriguing observation that SFFV can transform non-erythroid cells if it is co-expressed with sf-Stk. This results in transformation of fibroblasts in vitro and a variety of tumors in mice. We are currently utilizing the SFFV/sf-Stk-transformed fibroblasts to screen potential small molecule inhibitors for sf-Stk. We further showed that the human counterpart of sf-Stk, sf-RON, is expressed in certain types of human cancers, particularly ovarian and prostate cancers. Future work is aimed at determining whether sf-RON plays a causal role in these cancers and whether sf-RON, like sf-Stk, is activated by interacting with another protein, such as the envelope protein of a human retrovirus, to cause cancer. As a second retroviral model system, we have been studying PVC-211 murine leukemia virus (MuLV), a variant of the erythroleukemia-inducing Friend MuLV that causes a rapid neurodegenerative disease in rodents. The diseased brains and spinal cords of PVC-211 MuLV-infected rats exhibit the spongiform pathology characteristic of some human neurodegenerative diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis and transmissible spongiform encephalopathy. We previously demonstrated that subtle changes in the envelope gene of the virus altered its host range from that of its leukemia-inducing parent Friend MuLV, enabling it to gain access to the central nervous system by infecting brain capillary endothelial cells (BCEC). By comparing BCEC from uninfected and PVC MuLV-infected rats, we found that virus-infected BCEC express high levels of inducible nitric oxide synthase and show evidence of NO production. We recently demonstrated that PVC-211 MuLV infection of BCEC in vivo results in the production of vascular endothelial cell growth factor and the chemokine MIP-1alpha, leading to vascular leakage and activation of microglia, the resident macrophages of the central nervous system. Further studies demonstrated that depletion of microglia from rat brains blocks neurodegeneration induced by PVC-211 MuLV and that treatment with antiserum to MIP-1alpha or splenectomy, both of which reduce the number of activated microglia in the brain, can delay disease, clearly demonstrating the importance of activated microglia in the development of PVC-211 MuLV-induced neurodegeneration. Current studies are focused on using pharmacological inerventions to block or mitigate PVC-211 MuLV-induced neurodegeneration. We are currently using knowledge and reagents obtained from working with mouse retroviruses to study the xenotropic MuLV-related human retrovirus XMRV, which was recently discovered through an association with prostate cancer. In collaboration with the laboratories of Judy Mikovits and Frank Ruscetti, we were able to use antibodies developed against the envelope protein of SFFV to detect infectious XMRV in the blood cells and plasma of patients suffering from the neuroimmune disease chronic fatigue syndrome (CFS). We were further able to develop a seroconversion assay using cells expressing the SFFV envelope protein to detect antibodies against the virus in the plasma of CFS patients. We now plan to apply our knowledge of the pathogenesis of mouse retroviruses that cause cancer and neurological disease in rodents to study the molecular basis for similar diseases associated with XMRV. We are in the process of developing rodent models for determining the biological effects of XMRV in vivo, which if successful will provide a small animal model for preclinical testing of potential anti-XMRV drugs. In addition, we are testing both in vitro and in vivo the biological effects of the envelope protein of XMRV, which like its related SFFV counterpart may be responsible for the pathogenicity of XMRV.